Scl 10a hplc system

Lipid peroxidation was measured by MDA derivatization with TBA followed by malondialdehyde (MDA) derivatization with thiobarbituric acid (TBA) followed by high-performance liquid chromatography (HPLC) analysis [14 ]. The total MDA content was first released from cellular compartments in a mixture of 50 µL of plasma with 250 µL of 30% methanol for 15 min (4°C) in an ultrasound bath. Subsequently, 100 µL of 0.50% BHT was added to the samples to avoid oxidation reactions in the following steps. MDA was then converted into a pinkish chromophore by derivatization with 600 µL of 0.4% TBA (in 0.20 M HCl) for 30 min at 95°C, under constant mixing. The sample was then filtered (MilliPore nylon membranes, 0.45 µm pore size, 13 mm diameter) and injected (20 µL) in a Shimadzu SCL10A HPLC system equipped with LC10AD pumps and fluorescence (RF-10AXL) detector. The MDA-TBA adduct was isocratically eluted by a 65:35 mixture of 25 mM phosphate buffer (pH 6.5) and methanol 30% through a 0.39 × 30 cm µBondapack C18 column and detected by fluorescence (excitation/emission λ = 515/555 nm; retention time ∼ 6 min). The total MDA peak areas of the samples were compared with a standard curve obtained with 1,1,2,2-tetraethoxypropane (also in 30% methanol). Total MDA released in plasma was calculated by determining the area under curves between 0 and 60 min (AUC_0-60min).